Pre‐culture with transferrin‐Fe3+ before in vitro maturation improves the developmental competence of porcine oocytes matured in vitro

Abstract Purpose Since the developmental competence of oocytes cultured after in vitro maturation (IVM) is low, it is necessary to improve the IVM method for efficient offspring production. In this study, we revealed that transferrin (TF)‐Fe3+ was accumulated in follicular fluid with increasing the follicular diameter, and that TF receptor (TFR1) was localized in granulosa cells of pig. Thus, we hypothesized that TF‐Fe3+ would be a factor in the induction of developmental competence of porcine oocytes. Methods To mimic the follicular development environment, cumulus–oocyte complexes (COCs) were cultured in pre‐IVM medium (low dose of FSH) without or with Holo‐TF (monoferric or diferric TF) or Apo‐TF (non‐iron bond TF). After pre‐IVM without or with Holo‐TF, COCs were cultured in IVM medium (high dose of FSH and EGF) without or with Holo‐TF. Results Cultivation with Holo‐TF increased the expression of follicular development maker (Cyp19a1 and Ccnd2), E2 production, and proliferative activity of cumulus cells, whereas cultivation with Apo‐TF did not show these positive effects. The treatment with Holo‐TF during pre‐IVM, but not during IVM, dramatically induced oocyte maturation with increasing the blastocyst rate. Conclusion We succeeded in showing for the first time that the cultivation with Holo‐TF in pre‐IVM can produce embryos in pig with high efficiency.

ovulatory environment. Our previous reports showed that developmental competence of oocytes is improved by the cultivation in follicular development environment (pre-IVM), followed by the cultivation in ovulatory environment (IVM) with transition of hormone environment, including FSH, LH, E2, and P4. 4,5 Furthermore, other reports also showed that the preincubation before IVM of porcine COCs enhanced developmental competence of oocytes. [6][7][8] Therefore, it is effective that the cultivation of COCs with pre-IVM before IVM enhances developmental competence of oocyte. in granulosa cells, and these factors then induce COC expansion, oocyte meiotic maturation, and ovulation. [11][12][13][14][15] In pig, previous reports showed the effectiveness of FSH and EGF supplementation during IVM to induce nuclear maturation and cytoplasmic maturation of oocytes, although even today blastocyst production efficiency is below 20%. Since the dominant follicles are vascularized on the follicle surface, 5,16 we assumed that the blood-derived factor(s) enter follicle and induce full maturation in oocyte.
Transferrin (TF), a 78-kD glycoprotein, is secreted mainly from liver. 17,18 TF consists of two lobes: one at the N-terminal region and the other at the C-terminal region. 19 Since a lobe can bind to one iron, a TF molecule bind two ferric irons. TF-bound iron (TF-Fe 3+ ) circulates freely in the serum and in extravascular spaces, and it serves as a source of iron for cells and tissues that are perfused by the systemic circulation. TF receptor 1 (TFR1) functions as a dimer, and each 90-kD monomer has a single transmembrane-spanning domain. Most cells modulate iron uptake by regulating the amount of TFR1. 20 When TFR1 binds to TF-Fe 3+ , the TF-Fe 3+ -TFR1 complex is internalized into an endosome, where acidification facilitates the release of Fe 3+ from TF; the released Fe 3+ is used in various peripheral tissues, including muscle, heart, kidney, and bone marrow. 21 During follicular development, although the existence of TF protein was previously shown in granulosa cells, 22 the physiological effectiveness of TF on COC function has not been reported.
The aim of this study was to characterize the roles of TF-Fe 3+ in cumulus cells function and in the developmental competence of oocytes cultured prior to IVM (pre-IVM) followed by IVM with a physiological concentration of TF-Fe 3+ in pig. We firstly investigated the expression of Tf and Tfr1 mRNA in cumulus cells of COCs and the TF concentration in follicular fluid collected from follicles of various sizes. We then examined the effect of supplementation with TF-Fe 3+ complex to pre-IVM medium and/or IVM medium in which COCs were cultured on the function of cumulus cells and on developmental competence in pigs.

| Materials
tomycin (Nacalai Tesque), and ultrapure water. Modified NCSU37 (mNCSU37) medium was used as basic medium for pre-IVM and IVM of porcine COCs. 5,23,24 Highly purified FSH was a kind gift from the National Hormone and Pituitary Program (National Institute of Diabetes, Digestive and Kidney Diseases; NIDDK). Hypoxanthine (Sigma-Aldrich) was dissolved in the mNCSU37. Holo-TF (monoferric or diferric TF; T8158) and Apo-TF (non-iron bond TF; T2252) were purchased for Sigma-Aldrich and were dissolved in the mNCSU37.
Fetal calf serum (FCS) was purchased from Invitrogen.

| Isolation of porcine COCs and follicle fluid
Porcine ovaries and liver were collected from 5-7-month-old prepubertal gilts at a local slaughterhouse and transported within 1 h to the laboratory in 0.85% NaCl containing 1% penicillin-streptomycin mixed solution (Nacalai tesque) at about 30°C. Using a previously described methods, 5 follicles with diameters of small (1-3 mm), medium (4-7 mm), and large (bigger than 8 mm) antral follicles in the diameter were cut with scissors and were used for immunofluorescence staining. COCs were aspirated from each size follicles by an 18G needle and placed in a 1.5 mL tube. The tube was centrifuged at 100 g for 5 min, and the supernatant was stored at −80°C until used for the analysis of TF and iron concentration. Precipitates containing granulosa cells and COCs were placed in a petri dish, and COCs surrounded by more than four layer of unexpanded cumulus cells were collected with a Pasteur pipette under a stereomicroscope.
Collected COCs were used for total RNA isolation, pre-IVM culture, and IVM culture.

| Real time RT-PCR analysis
Total RNA was isolated according to our previous report. 5 Briefly, total RNA was extracted from COCs and liver using the RNeasy Mini Kit (QIAGEN Sciences) according to the instruction manual and dissolved in nuclease-free water. The final RNA concentration (5 ng/ μL) was determined by absorbance using a microspectrophotometer (Thermo Fisher Scientific).
Reverse transcription was performed as recently described. 16 Briefly, isolated total RNA (5 ng/μL) was reverse-transcribed using 500 ng of poly-dT (Promega) and 0.25 U of avian myeloblastosis virus reverse transcriptase (Promega) for 75 min at 42°C and for 5 min at 95°C.
Real-time PCR analysis was performed as we described recently. 16 Briefly, cDNA and primers were added to the KAPA SYBER FAST Universal qPCR kit (Kapa Biosystems) to give a total reaction volume of 15 μL. PCR was then performed using the MiniOpticon Real-Time PCR Detection Systems (Bio-Rad). The conditions were as follows: 30 s at 95°C, followed by 40 cycles each of 5 s at 95°C and 45 s at 60°C or 64°C. Specific primers were selected and analyzed as indicated in Table 1. 18s was used as a control for reaction efficiency and variations in concentrations of mRNA in the original RT reaction. In this study, several housekeeping genes such as 18s and Gapdh were tested. 18s showed the least variation among treatment groups and was closed to target gene; thus, 18s was used as the housekeeping gene in this study (data not shown). The results were first normalized to the expression levels of 18s. To avoid false-positive signals, dissociation curve analysis was performed at the end of amplification and the PCR products were applied to agarose gel electrophoresis to confirm the size.

| Measurement of iron concentration in follicular fluid
The fluids collected from small, medium, and large antral follicles were each treated with 6 M HCl to adjust the pH (2, 3), and were put under room temperature for 30 min. Each solution was then centrifuged for 10 min at 1000 g and the iron concentration of each supernatant was measured using an iron assay kit (Metallogenics, Co., Ltd.) according to the manufacturer's instructions.

| Measurement of E2 concentration
Extraction and E2 measurement were performed according to the manufacturer's instruction for the Estrogen ELISA kit (Cayman Chemical). In detail, pre-IVM culture medium and methanol were mixed at 1:4 and incubated for 10 min at room temperature. After incubation, the solution was centrifuged for 10 min at 2000 g and the supernatant was evaporated. The residue was reconstituted in ELISA buffer. E2 measurement was performed with the reconstituted solution using VARIOSKAN FLASH (Thermo Electron Corporation).

| Assessment of nuclear maturation
Oocytes were separated from cultured COCs, and the nuclear status was assessed as described previously. 24

| Pre-IVM and IVM of porcine COCs
For the pre-IVM and IVM of COCs, oocytes having an evenly granulated cytoplasm with at least four layers of unexpanded cumulus cells were selected by microscopic examination and washed three times. Twenty COCs were cultured in 100 μL of mNCSU37 medium at 39°C in a humidified incubator (95% air, 5% CO 2 ). The mNCSU37 medium was supplemented with 2 mM hypoxanthine, 10% (v/v) FCS (Gibco BRL) and 7 mM taurine (Sigma-Aldrich) and used as basic medium for pre-IVM and IVM.
In this study, we detected that TF concentration in follicular fluid collected from large antral follicle was 13.5 mg/mL. In 10% FCS of mNCSU37 medium, 0.5 mg/mL TF was included (data not shown), thus, we added the 13.0 mg/mL of Holo-and Apo-TF to pre-IVM medium and IVM medium in this study.
To investigate the effects of Holo-TF during pre-IVM on the cumulus cells functions of COCs and meiotic resumption of oocytes, COCs were cultured in Control, Holo-TF, and Apo-TF group for 6, 10, or 12 h. Control group was cultured in maturation medium supplemented with low dose (100 ng/mL) of highly purified FSH. Holo-TF or Apo-TF group were cultured in pre-IVM medium supplemented with low dose (100 ng/mL) of highly purified FSH and either 13 mg/ mL Holo-TF or 13 mg/mL Apo-TF ( Figure 1). After pre-IVM, culture medium was used for E2 concentration (12 h Holo-TF or Apo-TF group were cultured in IVM medium supple-

| In vitro fertilization (IVF)
In vitro fertilization was performed according to our previous report. 5 Briefly, cumulus cell-free oocytes were washed three times with fertilization medium, that is, modified Tris-buffered medium

| In vitro culture (IVC)
After sperm-oocyte coincubation, putative zygotes were cultured according to our previous report. 5  were fixed and placed on slides with drops of mounting media with DAPI (C-C: n = 60, H-C: n = 58).

| Statistical analyses
All data were obtained from three or four replications for compari-  follicle (small: 64.31 ± 5.26, medium: 73.97 ± 8.77 μg/dL, and large: 99.15 ± 9.96 μg/dL; Figure 2C). TFR1 protein localization in small and large antral follicles was low level, whereas the red signal showing TFR1 protein localization in medium follicles was higher, and the signals were in both granulosa cells and cumulus cells (Figure 3).

| Effect of pre-IVM with Holo-TF on meiotic resumption of oocytes, expression on follicular development makers in cumulus cells and E2 concentration in cultured medium after pre-IVM
During the follicular development, meiotic resumption of oocytes was suppressed by hypoxanthine, a physiological inhibitor of oocyte meiotic resumption in pigs. 28 Our previous study showed that the addition of 2 mM hypoxanthine significantly suppressed spontaneous oocyte meiotic maturation in pigs 29 ; therefore, in this study, COCs were cultured in pre-IVM medium supplemented with 2 mM hypoxanthine for 12 h, and the effect of pre-IVM on meiotic resumption of oocytes was investigated. The GVBD rate of oocyte cultured in control was almost 10% and the value was similarly low even when Holo-TF or Apo-TF was added to control medium ( Figure 4A). These low GVBD rates in control, Holo-TF, and Apo-TF groups were similar values to previous reports. [28][29][30] To mimic the follicular development environment, COCs were cultured in pre-IVM medium without (Control) or with Holo-TF for Holo-TF were significantly higher than those of 0 h and the Control.
Cultivation with Apo-TF significantly reduced these high Cyp19a1, and Ccnd2 mRNA expression levels obtained by cultivation with Holo-TF ( Figure 4B,C). The E2 concentration in follicular fluid of the Holo-TF treatment group was higher than that in Control (Holo-TF: 6.15 ± 0.96 ng/mL, Control: 0.83 ± 0.01 ng/mL); however, Apo-TF cultivation significantly reduced the E2 concentration in the Holo-TF group ( Figure 4D).

| Effect of pre-IVM with Holo-TF on proliferation of cumulus cells of COCs
In this experiment, we examined the effect of pre-IVM with Holo-TF

| Effect of IVM with Holo-TF on COC expansion, oocyte maturation, and oocyte developmental competence of COCs
To examine whether Holo-TF is necessary for COC expansion, oocyte maturation, and the developmental competence of oocytes of COCs during IVM, COCs were cultured in pre-IVM medium without or with Holo-TF or Apo-TF for 12 h, after which the cultured COCs were cultured in IVM medium without or with Holo-TF or Apo-TF for 3 h. Expression levels of Has2 and Ptx3 mRNA did not differ significantly among the treatment groups, whereas Tnfaip6 mRNA expression was significantly increased by cultivation in C-C, H-C, and H-A treatments compared with that in before culture (0 h; Figure 6A).
In the C-C group, the outer layer of the cumulus cells of COCs was expanded, however, the inside of the cumulus cells was not fully expanded. In the H-C group, both the outer and inner layers of the cumulus cells were fully expanded; however, the A-C treatment induced the expansion of only the outer layer of the cumulus cells. In the H-H and H-A groups, cumulus expansion was also not fully induced ( Figure 6B). After pre-IVM, COCs were cultured in IVM medium at Control, Holo-TF, and Apo-TF group and the degenerate rate, GV rate, GVBD rate, and MII rate was measured. The degenerate rate, GV rate were low in all groups, whereas GVBD rate in H-H group and H-A group were significantly high compared with that in C-C, H-C, and A-C groups ( Figure 7A-C). In the C-C, H-C, A-C, and H-A groups, the MII rate were relatively high, almost 80%; however, the rate in the H-H group was significantly lower by comparison ( Figure 7D).

Cumulus expansion index in
After insemination, the fertilization rate in the C-C and H-C group was almost 40%, and the blastocyst rate in the C-C group was low, but H-C treatment significantly and dramatically increased the rate (C-C; 14.4 ± 3.2% vs. H-C; 48.7 ± 3.9%; Figure 8A,B).

F I G U R E 4
Effects of pre-IVM with Holo-TF on GVBD rate, the expression of Cyp19a1 and Ccnd2 in cumulus cells, and E2 concentration in cultured medium. COCs collected from medium antral follicles (4-7 mm) were cultured in pre-IVM medium without or with Holo-TF or Apo-TF for 6 h (Cyp19a1 and Ccnd2) or 12 h (GVBD rate and E2). After cultivation, GVBD rate of precultured oocytes (A) was assessed, and expression of Cyp19a1 (B) and Ccnd2 (C) mRNA in precultured cumulus cells were analyzed by real-time RT-PCR. E2 concentration (D) in preculture medium was analyzed by ELISA. 0 h; Cyp19a1 or Ccnd2 mRNA expression in COCs just after collected from medium antral follicles. Control; COCs were cultured without TF. Holo-TF; COCs were cultured with Holo-TF. Apo-TF; COCs were cultured with Apo-TF.

| DISCUSS ION
Oocyte maturation occurs during two different phases: the follicular developmental phase and the ovulatory phase. In the former, COCs proliferate and accumulate the energy and nutrition needed for embryogenesis. 31,32 In the later, cumulus expansion and meiotic maturation of oocytes occur. 33 It is well recognized that matured oocytes formed through these two phases have high developmental competence. 4,34,35 COCs cultured by the IVM are usually recovered from developing follicles (4-7 mm diameter), however, COCs are conventionally cultured in the high dose of FSH condition where the ovulation phase during IVM is mimicked. [36][37][38][39] Thus, it is reasonable to adapt a novel IVM method that is consistent with the environment of both the follicular development phase and the ovulatory phase.
Although there are few reports about TF expression in the ovary, the localization of TF has been observed in granulosa cells in antral follicles during follicular development. 22 In this study, we also showed the expression of Tf mRNA in developing antral follicles, though levels extremely low compared to those in liver. On the other hand, we showed for the first time that TF concentration in large antral follicles was 13.5 mg/mL, more than threefold the approximately 4 mg/mL found in both serum and liver. 40 Since we confirmed that Tf mRNA, TF protein expression, and iron content in liver were significantly upregulated after eCG injection in mice (data not shown), we inferred that TF-Fe 3+ was supplied by the liver via blood vessels to induce follicular development. In hepatocytes, TRF1 is known to be recycled when it binds to TF, 41  Numerous studies showed that FCS supplementation to conventional IVM medium enhanced oocyte maturation during conventional IVM in mice, 42 rabbits, 43 bovine, 44 and pigs. 45   is not known whether this TF concentration is optimal for oocyte maturation. In this study, since the information of TF and iron concentration was collected from pig killed in slaughterhouse, hormonal (eCG and hCG) treatment did not conduct. However, our preliminary experiments using mice revealed that the eCG-primed mice ovaries showed twofold increase compared with non-eCG-primed mice ovaries (unpublished data). Moreover, we also showed that the TF in the liver and iron concentration in the blood was increased in response to eCG injection to mice (unpublished data). Collecting the above, it is the possibility that much higher concentration of TF contain in the preovulatory follicle.
To supply TF-Fe 3+ during pre-IVM, culturing with Holo-TF to pre-IVM medium, but not in IVM medium (H-C) was significantly F I G U R E 7 Effects of IVM with Holo-TF on the oocyte maturation. COCs collected from medium antral follicles (4-7 mm) were cultured in pre-IVM medium without or with Holo-TF or Apo-TF for 12 h and then were cultured in IVM medium without or with Holo-TF or Apo-TF for 48 h. Nuclear status of oocytes was evaluated by chromatin configurations ((A) Degenerate rate; (B) GV rate; (C) GVBD rate and (D) MII rate). 0 h; Has2, Ptx3, or Tnfaip6 mRNA expression in COCs just after collected from medium antral follicles. C-C; COCs were cultured in pre-IVM medium without TF, and then cultured in IVM medium without TF. H-C; COCs were cultured in pre-IVM medium with Holo-TF, and then cultured in IVM medium without TF. A-C: COCs were cultured in pre-IVM medium with Apo-TF, and then cultured in IVM medium without TF. H-H; COCs were cultured in pre-IVM medium with Holo-TF, and then cultured in IVM medium with Holo-TF. H-A; COCs were cultured in pre-IVM medium with Holo-TF, and then cultured in IVM medium with Apo-TF. Values are mean ± SEM of three independent experiments (n = 3). a-c, Significant differences were observed among each culture group (p < 0.05). COCs, cumulus-oocyte complexes; IVM, in vitro maturation; TF, transferrin.
improved COC expansion and developmental competence of oocytes compared to those in the C-C and A-C groups. Surprisingly, the continuing addition of Holo-TF during both pre-IVM and IVM cultures caused declines in both the COC expansion and fertilization rate. Furthermore, we also revealed that the supplementation of Holo-TF to maturation medium (without FSH) significantly suppressed spontaneous maturation of porcine oocytes (data not shown). These results implied that iron supplied from TF is necessary in the follicular developmental phase only, and not in the ovulatory phase. In other tissues, previous studies showed that a sufficient amount of iron promotes various cell functions 50,51 ; however, excess iron amount causes cell dysfunction by generating reactive oxygen species (ROS). 52,53 In the ovary, other studies showed that ROS represses COC expansion, oocyte maturation, and oocyte developmental competence. 54,55 Thus, it is possible that excess iron applied from TF may affect oocyte maturation.
In conclusion, we showed for the first time that TF and iron ac-

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this report.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data presented in this study are available on request from the corresponding author.

A N I M A L S TU D I E S
The study was conducted in accordance with the Guideline for the

Care and Use of Laboratory Animals of Prefectural University of
Hiroshima (approval number 18AO-006).

Masayuki Shimada
https://orcid.org/0000-0002-6500-2088 Yasuhisa Yamashita https://orcid.org/0000-0002-8187-1435 F I G U R E 8 Effect of pre-IVM with Holo-TF on fertilization rate (A) and blastocyst rate (B). COCs collected from medium antral follicles (4-7 mm) were cultured in pre-IVM medium without or with Holo-TF for 12 h, and then were cultured in IVM medium without Holo-TF and Apo-TF for 48 h. After cultivation, sperm and oocytes were coincubated in IVF medium, and then cultured for 7 days. C-C; COCs were cultured in pre-IVM medium without TF, and then cultured in IVM medium without TF. H-C; COCs were cultured in pre-IVM medium with Holo-TF, and then cultured in IVM medium without TF Values are mean ± SEM of three independent experiments (n = 3). * Significant differences were observed (p < 0.05). COCs, cumulus-oocyte complexes; IVM, in vitro maturation; TF, transferrin.